Standard curves radioimmunoassay procedure

The most common, but by no means the only or even the most promising, immunochemical assay for small molecules is radioimmunoassay (R1A). As an overview, an immunoassay involves chemically attaching the small molecule of interest (or a derivative of it) to a carrier protein and raising specific antibody titers to it in the serum of an animal. Very dilute antibody solutions are then used to bind the small molecule which has been radiolabeled. The competition of varying known concentrations of unlabeled material is measured and the resulting standard curve used to determine unknown concentrations (Table 1). The steps leading to the development of an R1A are outlined below followed by a description of other immunochemical procedures and an analysis of the attributes and limitations of immunoassay. 

In this paper, we will review our previous research on a radioimmunoassay (RIA) procedure for the detection and quantitation of picloram using polyclonal antisera (15). Furthermore, we will also discuss our research on indirect EIA procedures using monoclonal and polyclonal anti-picloram antibodies which were compared in terms of the characteristics of the standard curves and performance based on the determination of picloram in fortified water, soil extracts, plant extracts, and human urine samples (16). 

If the antibody is immobilised on Sepharose , the supernatant containing the free, radioactive peptide can be separated easily and assayed in a gamma counter. With a standard curve drawn for known amounts of peptide subjected to assay under exactly the same conditions, unknown amounts of peptide can be determined by interpolation on the standard curve. There are two potential problems with this type of radioimmunoassay. First, the peptide to be assayed perhaps does not contain Tyr. If it contains His, however, this may suffice since His can be iodinated, especially by an enzymic procedure described below. Alternatively, the peptide is allowed to react with the Bolton and Hunter reagent (Bolton and Hunter, 1973), prepared by iodina-tion of the ester of 3-(4 -hydroxyphenyl)propionic acid and /V-hydroxysuccinimide. Any free amino group can be acylated by this reagent. Secondly, reaction of a peptide with Nal and chloramine-T can cause oxidation of Met, Cys and even Tyr residues, which can interfere with complexation of the iodinated peptide with antibodies raised to the un-iodinated peptide. An alternative method (Holohan et al., 1973) of iodination uses lactoperoxidase in the presence of H202. As pointed out above, this procedure is applicable to the iodination of His residues. This method avoids modification of the side-chains of Met, Cys and Tyr. 

This relationship is established by measurement of samples with known amounts of analyte (calibrators). One may distinguish between solutions of pure chemical standards and samples with known amounts of analyte present in the typical matrix that is to be measured (e.g., human serum). The first situation applies typically to a reference measurement procedure, which is not influenced by matrix effects, and the second case corresponds typically to a field method that often is influenced by matrix components and so preferably is calibrated using the relevant matrix. Calibration functions may be linear or curved, and in the case of immunoassays often of a special form (e.g., modeled by the four-parameter logistic curve) This model (logistic in log x) has been used for both radioimmunoassay and enzyme immunoassay techniques and can be written in several forms as shown (Table 14-1). Nonlinear regression analysis is applied to estimate the relationship, or a logit transforma-... 

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